Phenolics Quantitative and their Chemical Fingerprint Analysis in Peanut Shells

To control the quality of peanut shells, a simple and reliable method by high-performance liquid chromatography (HPLC) was developed for phenolics quantitative determination and fingerprint analysis in 26 peanut cultivars. The ultrasonic extraction process of phenolics was firstly optimized by using 70% ethanol, a solid-liquid ratio of 1:15, and 3 extracted cycles of 50 min each. Three phenolics, including luteolin, eriodictyol, and 5, 7-dihydroxychromone were quantified, with ranges of 0.988-4.688, 0.688-2.425, and 0.313-1.038 mg/g, respectively. Total of three phenolics in Shuang-ji 2, Xiang-hua 509-77 and Qing-lan 8 cultivars were 7.851, 7.638, and 7.514 mg/g, respectively, much higher than that in other cultivars. In the fingerprint analysis, a consensus fingerprint with fourteen common characteristic peaks was identified, and the chromatogram similarity values were all over 0.936. Based on these results, the quality of peanut shell used as herb or raw material of Chinese medicine can probably be controlled by a qualification test which was composed of simultaneous quantitative determination of phenolic compounds and fingerprint and similarity analysis.


Introduction
Peanut is one of the top five oil crops in the world, is also the major oil and cash crop in China. There are more than three thousand peanut cultivars in China, hundreds of which are cultivated on a large-scale [1]. The peanut shell, a byproduct of oil production, was formerly used as animal feed in China. However, it was actually a traditional Chinese herb, and recently it has been used for drug production, as raw material of "Maishu Jiaonang", a blood-fat decreasing Chinese medicine in clinical practice. Previous studies have reported that the physiological activities of the peanut shell are closely related to its phenolic compound content. Peanut shells containing higher quantities of phenolic compounds were shown to be more effective as Chinese herbs [2][3][4]. Therefore, it would be beneficial to develop a rapid method for the separation and quantification of the main phenolic compounds in peanut shells. Peanut shells are rich in phenolic compounds and are safe for use in clinical treatment [5]. The compounds luteolin, eriodictyol, and 5,7-dihydroxychromone have been screened and identified as the main components in peanut shells by the DPPH-HPLC-DAD-TOF/MS method [6]. Luteolin is the most representative bioactive compound in peanut shells. It has become popular as a primary component and is used extensively in synthetic drugs and health products [2].
Furthermore, the bioavailability of luteolin in peanut shell extract is significantly greater than that of pure luteolin [7], and its content varied wildly in different cultivars, as shown in an analysis of 42 peanut cultivars from Korea [8]. Therefore, it is important to screen for peanut shells with higher phenolic content for their application. There have been reports about the quality control of peanut shells, but most are limited to a qualitative analysis of the phenolic compounds or a quantitative analysis of luteolin rather than a chemical fingerprinting study of the peanut shell [9,10].
There is a great diversity of peanut cultivars in China, so it is meaningful to study phenolic compounds from peanut shells of different cultivars and also the chromatographic fingerprint of them. The current study aimed to adequately extract phenolic compounds from peanut shells and simultaneous determine the main phenolic compounds in the shells of 26 peanut cultivars in China and to develop a characteristic fingerprint, which combined with quantitative analysis, to control the quality of peanut shell used as herb or raw material of Chinese medicine.
Chemicals and Reagents: 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Fresh DPPH stock solution (5 mM) was prepared by dissolving DPPH in methanol prior to the analysis. Reference compounds, including luteolin, eriodictyol, and 5,7-dihydroxychromone, were purchased from Shandong Engineering Technology Research Center (Jinan, China) with purities of over 98%. HPLC-grade methanol was purchased from Shandong Yuwang Group (Yucheng, China). Water for the HPLC analysis was purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA). All other chemicals and solvents used were of analytical grade.

Optimization of Extraction Conditions of Phenolic
Compounds from Peanut Shells: Three solvents (100% methanol, 70% ethanol and 60% acetone) were chosen for crude phenolic compound extraction. The extraction conditions were that approximately 1.0 g of peanut shell powder was immersed in 30 mL of solvent and ultrasonicated for 40 min at 40 kHz, 120 W, and 55 °C using XO-SM100 Ultrasonic microwave reaction system (Nanjing Atpio Instruments Manufacturer, Nanjing, China). After cooling to room temperature, made up the solvent to the original weight and centrifuged at 4000 rpm, 4 °C. The supernatants were collected for crude extracts. The respective extraction rates of luteolin, eriodictyol, and 5,7-dihydroxychromone and the DPPH scavenging activity of the crude extracts were taken as indices [6]. The ultrasonic extraction conditions were then optimized by the orthogonal array design (OAD) method. An L9 (34) orthogonal table was used to carry out an optimization experiment, and four important factors were set as follows: a) Ethanol concentration (60%, 70%, and 80%) b) Solid-liquid ratio (1:10, 1:15, and 1:20, w/v) c) Extraction cycles (1, 2, and 3); D: extraction time (30, 40, and 50 min). Finally, the extracted solutions were centrifuged at 4000 rpm at 4 °C, and the supernatants were collected for further use. Total yield of luteolin, eriodictyol, and 5,7-dihydroxychromone in the supernatants was taken as an index.

Quantification of Phenolic Compounds from Peanut
Shells by HPLC-DAD: Chromatographic analysis was performed on an Agilent 1200 series high performance liquid chromatography (HPLC) system (Agilent, Palo Alto, CA, USA) equipped with a diode array detector (190-400nm), a quaternary solvent delivery system, a column temperature controller and an autosampler. The chromatographic data were processed with Agilent Chromatographic Work Station software. HPLC conditions and standard curves of luteolin, eriodictyol, and 5,7-dihydroxychromone were determined as described by Qiu et al. [6], carried out at 30 °C on an Agilent ZORBAS SB-C18 reserved-phase column (250 mm×4.6 mm i.d., 5 µm), with a flow rate of 1.0 mL/min, and a linear gradient elution of eluents A (water-acetic acid, 100:0.2, v/v) and B (methanol) was used for separation, the elution program was shown in Table 1 the fractions were monitored at 294 nm. A quantitative analysis of luteolin, eriodictyol, and 5,7-dihydroxychromone in the shells of 26 peanut cultivars was carried out by an external standard method. Five different concentrations of standard mixtures according to levels estimated in the samples were injected for the calibration curve establishment, and calibration curves were constructed from peak areas versus compound concentrations. The concentrations of luteolin, eriodictyol, and 5,7-dihydroxychromone in the extracts were calculated by corresponding calibration curves and finally converted into content values for the raw peanut shell powders. Each sample was analyzed in triplicate to determine the mean content (mg/g).

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complete chromatographic profiles of the samples were calculated, the simulative median chromatogram was also calculated and generated, and the similarities of different chromatographic patterns were compared with the median chromatogram. In addition, the relative retention time (RRT) and relative peak area (RPA) of each characteristic peak relative to the reference peak were also calculated for quantitative expression of the chemical properties shown in the chromatographic pattern of the peanut shells.

Results and Discussion
Organic solvents combined with ultrasonic methods have been proven to be efficient for phenolic extraction [17][18][19] and were both optimized here for phenolic extraction from the peanut shell. The extraction efficiency of different organic solvents, including methanol, ethanol and acetone were firstly compared. The respective extraction rates of luteolin, eriodictyol, and 5,7-dihydroxychromone from peanut shells and the DPPH scavenging activity of the crude extracts were determined ( Figure  1). It was shown that the extraction rates of phenolic compounds and DPPH scavenging activities in 70% ethanol and 60% acetone were significantly higher than that in 100% methanol. Finally, ethanol was selected as the extraction solvent because there was no significant difference between 70% ethanol and 60% acetone (P>0.05), and ethanol is more cost effective and less toxic than acetone.

Volume 11-Issue 1: 2018
Ultrasonic extraction conditions were optimized by the orthogonal array design (OAD) method, which has been commonly used in phenolic extraction [20,21]. Based on the results of single factor tests [22], a further optimization process using OAD method was carried out (Table 2). According to the range analysis, the effects of each factor on the extraction of luteolin, eriodictyol, and 5,7-dihydroxychromone in order were as follows: extraction cycles (C)>extraction time (D)>ethanol concentration (A)>sample-solvent ratio (B). According to variance analysis, the effect of factor C was significant (P<0.1), while the effects of factors D, A and B were not significant (P>0.1). Based on this analysis, the optimum extraction conditions were determined to be an ethanol concentration of 70%, a solid-liquid ratio of 1:15 (g/mL), 3 extraction cycles, and an extraction time of 50 min. And the amount of extracted total phenolics reached as high as 7.553 mg/g. The whole phenolic extraction process is simple, economical and feasible for industrial production.
Note: (A) ethanol concentration; (B) sample-solvent ratio; (C) extraction cycles; (D) extraction time; (ki) The average value of the experimental results corresponding to the horizontal number (i) of any column; (R) The maximum of ki value minus the minimum of ki value under each factor. The peanut shells of 26 peanut cultivars (S1-S26) were extracted by the optimized ultrasonic method described above, and the contents of three main phenolic compounds (luteolin, eriodictyol and 5,7-dihydroxychromone) were simultaneously determined by HPLC. As shown in Table 3 luteolin, eriodictyol, and 5,7-dihydroxychromone were present in the shells of all 26 peanut cultivars, with content ranges of 0.988-4.688, 0.688-2.425, and 0.313-1.038 mg/g, respectively. The total quantity of the three main phenolic compounds in S16, S19 and S14 were, respectively, 7.851, 7.638, and 7.514 mg/g, much higher than in the other cultivars. To obtain a consensus chromatographic fingerprint, the HPLC fingerprints of 26 peanut shell samples were generated at the UV absorption of 294 nm (Figure 2).  The median chromatogram was regarded as the consensus fingerprint of peanut shells. The peaks present in all chromatograms of the samples were assigned as the "common peaks". Fourteen common characteristic peaks in the 26 chromatograms were selected (Figure 3), The retention times of these peaks were 10.37, 12.12, 12.79, 14.22, 19.50, 21.76, 26.41, 28.06, 31.15, 32.61, 34.51, 37.29, 40.42, and 50.27 min, respectively. Peak 11 (retention time=34.51 min, luteolin) was chosen as the internal reference peak to calculate the relative retention time (RRT) and relative peak area (RPA). The RRT and RPA of common peaks in the 26 samples are shown in Table 4. A similarity analysis of all of the peanut shell samples was conducted by comparison to the consensus fingerprint, and the results are shown in Table 5. The closer the cosine values are to 1, the more similar two chromatograms are. The similarity values of the 26 samples were all over 0.936, suggesting that the peanut shells had relatively similar chemical compositions.  The internal reference substance. The fingerprint pattern of different samples varies, but the similarity value of each fingerprint pattern could be obtained by comparing each fingerprint pattern with the median chromatogram, which could facilitate the evaluation of the quality of different samples. Based on our results, we could recommend a suitable similarity value in future qualification tests.

Conclusion
The results showed that the optimized ultrasonic extraction method is simple, economical and feasible. The contents of luteolin, eriodictyol, and 5,7-dihydroxychromone in the shells of 26 peanut cultivars in China were significantly different (P<0.05) between cultivars. Among them, total of three phenolics in cultivars S16, S19 and S14 were much higher than in the other cultivars, with values of 7.851, 7.638, and 7.514 mg/g, respectively. The result of fingerprint and similarity analysis suggested that the peanut shells had relatively similar chemical compositions. Based on above results, the quality of peanut shell used as herb or raw material of Chinese medicine can probably be controlled by a qualification test which was composed of simultaneous quantitative determination of phenolic compounds and fingerprint and similarity analysis by the lowest possible phenolic content and similarity value recommendation.